Process and composition for cleaning medical instruments

ABSTRACT

The invention relates to a method for cleaning a contaminated medical instrument including the step of immersing the instrument in a solution containing an enzyme based cleaning composition including a “hospital grade disinfectant”. Compositions useful for cleaning contaminated medical instruments in accordance with the method include an enzyme, a quat biocide and an “activity protector”, which may be for example, enzyme stabilizers, enzyme stabilizing systems, micelle formation modifiers and inhibitors, and combinations thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a 371 of PCT/AU01/00381, filed Apr. 5, 2001,which claims priority to Australian Patent Application No. PQ6791, filedApr. 7, 2000, both of which are incorporated by reference herein intheir entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] Not applicable.

REFERENCE TO MICROFICHE APPENDIX

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] This invention relates to a method of and composition forcleaning medical instruments. The method is suitable for use withendoscopes and is herein described with particular reference to thatuse, but it will be understood that the method is equally suitable foruse with other instruments such as colonoscopes, laparascopes, othersurgical, medical, biopsy, dental and such like instruments, parts ofsuch instruments and similar paraphernalia (hereinafter collectivelyreferred to as “medical instruments”). The invention is also applicablefor treatment of instruments which are required merely to be disinfectedfor example hair-dressing tools, certain beauty parlour equipment, andthe like.

BACKGROUND OF THE INVENTION

[0005] Endoscopes are increasingly being used in medical diagnosis andtherapy When used as directed the endoscope becomes grossly soiled andmassively infected with microorganisms which are present in bodycavities, on the mucous membrane, and in the blood. Accordingly theinstruments must be thoroughly cleaned and disinfected after each use.Endoscopes are precision instruments which are made from a combinationof materials. They are difficult to clean in view of the sensitivity ofthe materials involved to chemical attack and because they have narrowlumens making access to and cleaning of interior surfaces difficult.

[0006] Until the last decade, it was common for soiled instruments to beplaced on a towel or in a covered pan until they were sent to acentralised service where they were scrubbed, washed and eithersterilised in a steam autoclave (if not heat sensitive) or chemically(e.g. with formaldehyde). In the last decade, there has arisen aparticular concern to avoid transmission of very serious and sometimesfatal diseases such as may be carried in blood and tissue, for examplehepatitis B, HIV, and other infections.

[0007] Nowadays, contaminated endoscopes and other medical instrumentsare typically treated in a first bath (“presoak” or “cleaning” bath)containing one or a combination of anionic and non ionic surfactants.The first bath may optionally include one, or a combination of enzymes,adapted to digest biological contaminants including cellular material,blood and other body fluids. Enzyme containing pre-soaking liquids aresignificantly more efficient in removing water insoluble and proteinsoils and are now considered the industry standard. In the case ofsurgical instruments requiring to be sterilised, the instruments aretypically then removed from the first bath, washed free of enzymesolution and other residues, and then deposited in a second bathcontaining a chemical sterilizing agent (for example, glutaraldehyde).The first bath container is subsequently washed and then furnished witha fresh enzyme solution so that the process may be repeated. Thenecessity for separate cleaning and sterilizing baths arises sinceenzymes are denatured by all known sterilizing agents and sincesterilizing agents are deactivated by enzymes (as enzymes are proteins).Accordingly it has to date proved impossible to provide a “single bath”cleaning and sterilizing treatment, although a two part system involvingan enzyme treatment followed by addition of a phenolic disinfectant inthe same bath has been proposed.

[0008] Sterilisation protocols are followed to prevent cross infectionand therefore instruments used with one patient are not combined withthose which may have been used with another in the presoak bath. It isnoteworthy that the presoak is not passive. Staff are instructed tosyringe detergent liquor through all the lumens, to brush biopsychannels, etc. A colonoscope requires up to 14 manualbrushing-syringing-plugging-unplugging operations. It is usual for staffto wear latex gloves when handling instruments into or out of the bathsand when performing such like operations.

[0009] The present inventor has observed that currently used procedures,while effective for preventing crossinfection between patients, in factexposes medical and/or hospital staff to hitherto unrecognised healthand safety risks. By virtue that the enzymes of the first bath digestthe biological secretions holding the microorganisms, thus releasingthem within the bath, and surfactants efficiently disperse them, thefluid content of the first bath is itself readily contaminated to highlevels with infectious material. Contrary to the belief of some hospitalstaff, enzymes do not kill bacteria but rather release them. The presentinventor has measured bacterial counts in excess of 1×10⁹ colony formingunits (“cfu”) per sq. cm. on instruments entering the first bath

[0010] Staff are therefore at risk of infection (i) from splashes fromthe first bath either during scrubbing to release contaminants or duringdraining the first bath (or from splashes if an instrument isaccidentally dropped into the bath), (ii) from glove failures (latexgloves have a “pinhole” failure rate of about 12%), (iii) fromaccidental glove immersion above the wrist line, (iv) from finger stickincidents in the bath resulting in glove and sometimes dermalpenetration, (v) from aerosols created by brushes and syringes. Inaddition the wall surface of the first bath remains contaminated afterthe bath has been emptied and if not itself disinfected may be handledby unprotected staff. The last mentioned risk may be minimised byperforming the sterilisation step immediately after the digestion stepin the same container, but this does not avoid any of the other hithertounrecognised risks and is wasteful in use of excess sterilant.

[0011] In some cases instruments may not be required to be sterilised,for example with spatulas, and holders which do not penetrate the bodytissue, hair dressing implements and the like, it may be sufficient todisinfect the instruments to an appropriate standard. In such cases itwould be desirable to provide a cleaning and disinfecting treatmentcapable of meeting the required standards with a single composition.

[0012] Any discussion of the prior art herein is not to be construed asindicative of the state of the common general knowledge in the field.

[0013] It is the object of the invention to avoid or ameliorate theabove discussed disadvantages of prior art, or at least provide acommercial alternative to the prior art.

[0014] It is an object of preferred embodiments of the present inventionto avoid or at least ameliorate the risk of infection to personscleaning medical instruments by such procedures.

[0015] It is a further object of at least some of the preferredembodiments to provide a single step cleaning and disinfectingcomposition for use in cleaning medical instruments.

[0016] Preferred embodiments of the invention also address the risk ofcross infection of instruments by virtue of multiplication ofmicroorganisms, if any, which remain on the bath walls after each cycleof instrument cleaning.

[0017] It is an object of some embodiments of the invention to providesimple means for cleaning and disinfecting surfaces which require to bedisinfected

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

[0019] According to a first aspect the invention provides a method forreducing the risk to health care workers when cleaning a medicalinstrument contaminated with an organic load including protein, saidmethod comprising the step of treating the contaminated instrument witha liquid composition including a protease, a monoquaternary ammoniumdisinfectant and an activity protector, the activity protector being ofa kind and in a concentration selected so that the liquid provideshospital grade disinfection (as herein defined) in the presence of saidprotease and said load.

[0020] Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”.

[0021] In preferred embodiments of the invention the treating stepincludes immersion of the instrument in the solution and is followed bya second treatment in which the instrument is sterilised. However forsome disinfecting purposes it may be sufficient to rinse the instrumentwith sterile water after the first treatment or to provide a furtherdisinfecting treatment.

[0022] In Australia, disinfectants are graded according to testsspecified by the TGA in order of decreasing efficacy as Grade B“Hospital Dirty”, Grade A “Hospital Clean”, Grade C“household/commercial”. A copy of “The TGA Disinfectant Test” isannexed. The TGA tests are specified as TGO 54. Similar tests andclassifications are applicable in other countries. The term “hospitalgrade” disinfectant” is herein used to refer to disinfectants passingthe Grade A test, i.e. a “hospital grade” disinfectant must be at leastHospital Grade A. The TGA specification requires that a “hospital gradedisinfectant is able to give at least an 8 or 9 log reduction ininoculum density within 8 minutes

[0023] The applicant has discovered a method whereby a hospital gradedisinfectant, preferably a quaternary ammonium biocide (“quat. biocide”)can be included in a cleaning composition which in use comes in contactwith proteinacious material such as blood and other biologicalcontaminants such as are encountered in cleaning endoscopes and othersurgical instruments.

[0024] Hitherto it has been accepted that quat. biocides areinstantaneously deactivated by protein and certain ions such as thosefound in hard water. It is therefore surprising that a grade Adisinfectant can be effective in such an environment as is made possibleby the present invention. Even more surprisingly, in preferredembodiments of the invention the quat. biocide is in the form of aliquid concentrate which retains its biocidal activity in prolongedshelf-storage in contact with one or more enzymes which are alsoproteins which normally would be expected to quickly deactivate thequat. biocide. Surprisingly, also, the enzymes are not irreversiblydenatured. The liquid concentrate is readily diluted with water for use.

[0025] It will be appreciated that a first treatment according to theinvention does not result in disinfection or sterilisation of theinstruments and for that purpose a subsequent disinfection orsterilizing treatment may be required. However the presence of aneffective disinfectant in the enzyme bath is sufficient to preventmultiplication of microorganisms in the bath and to afford a degree ofprotection for staff not previously available from accidental infectionin the risk circumstances previously outlined.

[0026] According to a second aspect the present invention provides aliquid composition for use in cleaning a medical instrument contaminatedwith an organic load including a protein, said composition including:

[0027] a protease

[0028] a mono quaternary biocide; and

[0029] an activity protector of a kind and in a concentration selectedso that the liquid provides hospital grade disinfection (as hereindefined) in the presence of said protease and said load.

[0030] The “activity protector” is described more particularlyhereinafter

[0031] In highly preferred embodiments, compositions according to thesecond aspect also include a non-ionic surfactant;

[0032] Desirably the “activity protector” or “activity protector” systemis present in a concentration sufficient that the quaternary ammoniumbiocide is effective in use to provide “hospital grade A” disinfection(as herein defined) of the bath in the presence of the at least oneenzyme and of a typical proteinaceous load in the bath.

[0033] The “activity protector” is a composition selected from (1)compositions known to be effective in stabilizing enzymes in liquidaqueous solutions, including enzyme stabilizing compounds and systems,(2) selected “micelle inhibitors”, and mixtures of (1) and (2). Inpreferred embodiments of the invention the “activity protector” is anenzyme stabilizer and more particularly is a suitable concentration ofboron anions. Desirably these are solvated in a polyol and may becombined with enzyme stabilizing synergists or adjuvants forming anenzyme stabilizing system. Preferred “micelle inhibitors” includespecies known to modify as well as to inhibit micelle formation and maybe selected from water miscible solvents such as C1-C6 alkanols, C1-C6diols, C2-C24 alkylene glycol ethers, alkylene glycol alkyl ethers, andmixtures thereof. A highly preferred micelle inhibitor is di-(propyleneglycol) methyl ether.(“DPM”) and analogues thereof which modify micelleformation. It is especially preferred to combine the use of borate ionswith DPM which has been found by the present inventor synergistically toenhance the biocidal activity protection conferred on the quat. biocidewithout irreversibly denaturing the enzyme.

[0034] It is highly preferred that the quat biocide is an aryl quatcompound, preferably benzalkonium halide.

[0035] It is well known that enzymes may become denatured in storage, inthe presence of other enzymes, and/or in the presence of antagonisticanions such as for example anionic surfactants, quaternary ammoniumcompounds and detergency “builders”. A number of enzyme stabilizingsystems have been developed and are well known in the enzyme formulationart. An example of an “enzyme stabilizing system” is a boron compound(e.g. boric acid) which in the past has been used alone or with selectedother adjuvants and or synergists (e.g. polyfunctional amino compounds,antioxidants, etc) to protect proteolytic and other enzymes in storageand in various products. It has been theorised that an enzymestabilizing system such as boron and calcium form intramolecular bondswhich effectively cross-link or staple an enzyme molecule so as to holdit in its active spatial configuration. Enzyme stabilizers have nothitherto been used to protect the biocidal activity of a quat. biocide.The present invention is based on the surprising discovery that at leastsome enzyme stabilizing systems are effective in protecting the biocidalactivity of quat. biocides in the presence of protein.

[0036] In accord with the present invention the ratio of “activityprotector” e.g. boron to quat. biocide is preferably chosen tosubstantially to minimise the Minimum Inhibitory Concentration (“MIC”)of quat. biocide in the presence of the enzymes in the formulation andat a given level of protein load. MIC is a measure of the minimumconcentration of the biocide which succeeds in preventing bacterialgrowth in a culture during a specified time period, for example 24 hrs.Details of the MIC test are shown in “Bailey & Scott ‘DiagnosticMicrobiology’, 8^(th) edition, 1990 at page 177. The TGA tests arespecified at TGO 54 annexed. MIC tests referred to herein are conductedover 24 hrs

[0037] In the present case in which an enzyme is present in addition tothe quat. biocide and in which it is desired to retain the enzymaticactivity of the enzyme as well as the biocidal activity of the quat,biocide then the quantity of “activity protector” required will need tobe greater than that required merely to protect the enzyme and will needto be sufficient both to stabilise the enzyme and protect the biocidalactivity of the quat. biocide. Moreover as the composition isanticipated to come into contact with an external proteinaceous load(from contaminants in the surgical instruments bath) then the “activityprotector” concentration will need to be greater still.

[0038] The inventor has discovered that boron surprisingly protects aquaternary biocide from deactivation by a protein in such a way and tosuch an extent that the MIC of the biocide is not increased in thepresence of a protein. In preferred embodiments of the invention the MICis dramatically reduced, for example, more than halved notwithstandingthe presence of up to 2 wt. % based on the weight of solution, ofprotein. This allows the formulation of a wide range of new and usefulcompositions which remain effective as disinfectants or antibacterialsin circumstances in which the prior art would be significantly lesseffective or not effective at all.

[0039] The invention also enables storage-stable liquid biocidallyeffective compositions to be prepared with a lower concentration ofquat. biocide and at much lower cost. By “shelf stable” is meant thatthe composition retains at least 50% of its biocidal efficacy after 12months storage in a sealed container at 18-25° C. Preferred embodimentsof the invention retain better than 98% biocidal efficacy under theseconditions.

[0040] Without wishing to be bound by theory, the inventor speculatesthat polymeric borate ions associate with the cationic quat. biocide,thus protecting the quat biocide from combining with proteins. When theformulation is diluted the polymeric ions become unstable and releasethe quat biocide for disinfection. Alternatively, it may be that thebiocidal activity of the quat. biocide significantly relates todenaturing proteins of cell membranes and that boron complexes withcharged groups of non-living proteins and prevents wasting quat. ondenaturing non-living proteins. However as enzymes are structurallyquite different from quat. biocides, and as the complete mechanism bywhich quat. biocides kill bacteria is also uncertain, it was notpreviously predictable that any enzyme stabilizer would be effective inmaintaining the biocidal activity of a quat. biocide (an enzymeantagonist). The mechanism by which the activity of the quat biocide ismaintained may be different from that whereby the enzyme is stabilised.

[0041] Other “activity protectors” are discussed hereinafter.

[0042] According to a third aspect the invention provides a compositionaccording to the second aspect further including a nonionic surfactant.

[0043] Preferably the nonionic surfactant is one or a combination ofsurfactants selected from the group consisting of ethoxylates orpropoxylates and block copolymer of these.

[0044] A highly preferred embodiment of the invention, provides aneconomical effective cleaning and disinfecting composition whichcontains enzymes, is shelf stable in storage in concentrated form, isreadily diluted to a working concentration and remains biocidal in usein the presence of protein. Desirably the working dilution is effectivein use as a Hospital Grade A disinfectant.

[0045] The invention will now be more particularly described by way ofexample only with reference to various embodiments.

[0046] Example 1 is a composition which is a concentrate stable instorage but which in use is diluted with water from 200:1 to 1000:1(i.e. 200 or 1000 parts/wt water to 1 part/wt concentrate) Thecomposition is useful for comparing the effects of addition of variouscomponents to a Quat. biocide

EXAMPLE 1

[0047] g/l Benzyl dimethyl ammonium chloride, CAS 68424-85-1 150 Sodiumteraborate decahydrate, CAS 12007-42-0 30 Glycerin, CAS 56-81-5 25Terric GN9 (note 1) 200 Dipropylene Glycol Methyl Ether CAS 34590-94-8100 Water balance to 1000

[0048] Preparation

[0049] The sodium tetraborate is dissolved/suspended in the glycerol at80° C. The quaternary biocide and Terric GN9 (non-ionic detergent) arecombined with the DPM and the pH adjusted with e.g. acetic acid to pH7.2-7.3. The borate/glycerin solution is then combined with thequaternary biocide

EXAMPLE 2

[0050] Example 2 is identical to example 1 but includes 0.1% ofsubtilisin (0.1% protease enzyme).

[0051] Table 1 shows MIC results obtained with various combinations ofcomponents of example 1 and example 2 in the absence of the boron and,in accordance with the invention, in the presence off the boron. InTable 1 “Quat” is an abbreviation for benzyl dimethyl ammonium chlorideTABLE 1 MIC, ppm MIC, ppm composition (no boron) (with boron) A 1 quat(prior art) 20 12 2 quat + DPM 16 8 3 quat + GN9 25 8 4 quat + DPM + GN916 <8 B 1 quat + subtilisin (0.1% protease enzyme) 50 25 2 quat + DPM +enzyme 25 12 3 quat + GN9 + enzyme 25 12 4 quat + dpm + GN9 + enzyme 258

[0052] Table 1 part A compares the MIC of various quaternary ammoniumbiocidal compositions in the absence of boron and in the presence ofboron but in the absence of protein. MIC was measured by test methoddescribed in Bailey and Scott Diagnostic Microbiology, 8^(th) edition,1990, p.177 using one of the most resistant to QUATs strains ofPseudomonas aeroginosa ATCC No. 15442.

[0053] Table 1 exp. 3 shows that Terric GN9 deactivates the quat.biocide as would be expected. Unexpectedly, DPM enhances the activity ofa quat. even in the presence of GN9, while in each case the combinationwith Boron produces a marked improvement in biocidal efficacy incomparison with the combination in the absence of boron.

[0054] Table 1 part B shows the results in the presence of proteoliticenzyme subtilisin. As is apparent from the table the presence of theenzyme in the absence of boron results in a considerable reduction inefficacy of the disinfectant.

[0055] It is noteworthy that there is an improvement in efficacy of thequaternary biocide (reduction in MIC) in the presence of DPM even in thepresence of the deactivator Terric GN9. However the results aresignificantly better with boron than without. The combination of DPMwith boron synergistically improves the biocidal activity protection ofthe boron in comparison with compositions lacking boron or DPM (even inthe presence of Terric GN9) achieving an end result of higher efficacyin the presence of the enzyme and the Terric GN9 than obtainable withthe quat. biocide in the presence of 0.1% of subtilisin in the absenceof boron.

[0056] A preferred embodiment of the invention is shown in example 3.The composition of example 3 is a concentrate intended for dilution 1part/wt concentrate in 200 parts/wt water. The composition is intendedfor application as a pre-soak for surgical instruments.

EXAMPLE 3

[0057] Component % w/w A. Nonyl phenol ethoxylate (Terric GN9) 3 Di(propylene glycol) methyl ether 5 Perfume .1 Water 15 B. Sodiumtetraborate decahydrate 6 Glycerol 4 water 5 C. Acetic acid to pH 7.2 -7.3 D. Ethylene Glycol 5 (10% subtilisin) Alcalase 2.5 DL 3 E.Benzalkonium Chloride 80% 30 Water to 100

[0058] Premix Borax with hot water and glycerin, add to A, adjust pH,let the mixture cool down 30° C. and then slowly add premixedingredients D. Then add water premixed with Benzalkonium Chloride 80%.

[0059] The composition is stable for at least 1 year prior to use whenstored in a sealed container at 18-25° C. and when diluted with water200:1 is biocidally effective in use for at least 24 hrs. achieving“Hospital GRADE A” disinfection.

[0060] The composition is also biocidally effective at dilutions of lessthan 200:1, for example, 20:1.

EXAMPLE 4

[0061] Example 4. In-surgery comparison of detergent concentrates forpre-cleaning of flexible colonoscopes.

[0062] An in-vivo evaluation was performed in a surgery randomly over aperiod of nine months on 105 colonoscopes after colonoscopy procedures.

[0063] Colonoscopes were soaked in 10 L of diluted 200:1 concentrates ofthe following detergents:

[0064] 1. 15% sodium laurel sulphate +20% Dodecyl benzene sulphonic acidat pH=9

[0065] 2. 10% Dodecyl benzene sulphonic acid 10% Nonyl phenol ethoxylate(Terric GN9), 10% Alcalase 2.5 DL (subtilisin)

[0066] 3. Composition described in example 3.

[0067] Total soaking time—10 minutes. This was followed by rinsing with10 L of sterile distilled water.

[0068] The level of bacterial contamination of:

[0069] washing liquor—liquid in the bath (B)

[0070] biopsy channels (C)

[0071] surface of the endoscope (S)

[0072] were determined by plating of

[0073] the washing liquor (B),

[0074] 10 mL of water syringed through the biopsy channel (C)

[0075] and cotton swabs after wiping 5 sq. sm of the endoscope surface(S)

[0076] of serially diluted samples onto Macconkey agar plates.

[0077] The bacterial contamination of the same parts of colonoscope andthe washing liquor when instruments were soaked in sterile distilledwater were used as control.

[0078] Table 2. Total bacteria counts of the contaminated surfaces oncolonoscopes after soaking in detergents (statistically processedaverage of 105 samples, Statistical significance (t-test) was set atp<0.05): TABLE 2 B C washing Biopsy S liquor channel Surface 1 2.2 × 10⁹6.2 × 10³ 9.5 × 10³ 2 4.0 × 10⁹ 5.1 × 10¹ 2.9 × 10¹ 3 0 0 0 Control 3.7× 10⁸ 4.1 × 10⁶ 9.1 × 10⁴

[0079] This clearly indicates that concentrates that contain no quat.biocide only release (disperse) the bacteria and soils, creatingalarmingly high bacterial contamination in cleaning baths and leavingsignificant amount of viable bacteria on colonoscopes. Using the presentinvention, one achieves a safe working environment. In the case of aninstrument that is intended for contact with unbroken skin, theconcentrate may be used for a one-step cleaning disinfection procedure.

[0080] The Quat. Biocide

[0081] The invention has been exemplified by reference to alkyl benzyldimethyl ammonium chloride (also known as benzalkonium chloride) as thehighly preferred quat. biocide. However those skilled in the art willrecognise that other monomeric quaternary ammonium antimicrobialcompounds may be used.

[0082] It is preferred that the quaternary ammonium antimicrobialcompound is selected from the group having a general formula:

[0083] wherein R′ R″ R′″ R″″ are alkyl radicals that may be the same ordifferent, substituted or unsubstituted, branched or unbranched, andcyclic or acyclic. X is any anion but preferably a halogen, morepreferable chlorine or bromine.

[0084] Highly preferred antimicrobial compounds are mono-long chain,tri-short chain, tetralkyl ammonium compounds, di-long-chain, di-shortchain tetralkyl ammonium compounds and mixtures thereof, where by “long”chain is meant about C6-C30 alkyl, and by “short” chain is meant C1-C5alkyl, preferably C1-C3, or benzyl, or C1-C3 alkylbenzyl. Examplesinclude monoalkyltrimethyl ammonium salts such as cetyltrimethylammonium bromide (CTAB), monoalkyldimethylbenzyl compounds ordialkylbenzyl compounds. Quat. biocides such as chlorhexadine gluconatemay be employed.

[0085] The most highly preferred compounds for use in the invention haveat least one benzyl radical which may be a substituted benzyl. Examplesinclude C8-C22 dimethyl benzyl ammonium chloride, C8-C22 dimethyl ethylbenzyl ammonium chloride and di-C6-C20 alkyl dimethyl ammonium chloride

[0086] The quaternary ammonium compound is incorporated for broadspectrum (gram positive and gram negative) antibacterial properties andshould be present at least in an amount which would be effective forthat purpose in the absence of protein or other deactivator. It issurprising that compositions according to the invention have excellentshelf stability both in concentrated and dilute form.

[0087] Activity Protector

[0088] According to the invention the biocidal activity of thequaternary biocide is in use protected by an “activity protector” whichis a composition (an ion, compound, or combination thereof) selectedfrom the group of known “enzyme stabilizing systems” including bothreversible and irreversible enzyme inhibitors such as described in“Handbook of Enzyme Inhibitors”, Zollner H., 2^(nd) ed. VCH 1993. Thepreferred activity protector is a boron compound or more preferably amixture of a boron compound and a polyol. The boron compound may forexample be boric acid, boric oxide, borax, or sodium ortho-, meta-, orpyro-borate. In some formulations it may be desirable to use aperborate, such as sodium perborate to obtain a bleaching effect. Themost preferred boron source is sodium tetraborate. The protective effectof the boron compound may be enhanced by the presence of formate, orcalcium ion, or by polyfunctional amino compounds such as di- ortri-ethanolamine. Other activity protection enhancers, or adjuvants,include anions such as phosphates, citrates, sulphates and sequesteringagents such as used as water softeners such as EDTA.

[0089] In systems which use boron to stabilise enzymes the addition ofantioxidants and/or polyfunctional amino compounds has been reported toproduce a synergistic enzyme stabilizing effect and the use of suchenzyme stabilizer synergists in the present system is contemplated. Theterm “enzyme stabilizer systems” is used herein to denote combinationsof stabilizers with enhancers, adjuvants and/or synergists and the like

[0090] Polyol

[0091] The quat. biocide activity protector desirably includes a polyol.

[0092] The polyol is preferably one containing from 2-6 hydroxyl groupsand containing only C, H, and O atoms. Typical examples are ethyleneglycol, propylene glycol 1,2 propanediol, butyleneglycol and mostpreferably glycerol. Other polyols such as mannitol, sorbitol,erythritol, glucose, fructose, lactose, etc may also be useful. Thepolyol is selected to solvate the boron and increase its ionic strengthin the composition and will usually be present in an amount at leastequal to the amount of boron compound.

[0093] Micelle Inhibitors

[0094] A water miscible solvent is desirably included to assist insolubilising the components and/or substances with which the compositioncomes into contact depending on its intended use and avoid or inhibit ormodify micelle formation. This acts synergistically as an “activityprotector” as well as apparently in some instances enhancing biocidalactivity in its own right.

[0095] Preferably a water miscible solvent is selected from C1-C6alkanol, C1-C6 diols, C3-C24 alkylene glycol ethers, alkylene glycolalky ethers and mixtures thereof. A highly preferred solvent is di(propylene glycol) methyl ether. Other known micelle antagonists includeborates, lactates, citrates, tartrates. Enzymes

[0096] The boron stabilizer is added in an amount required to preventdeactivation of the enzyme in the presence of protein. Surprisingly ithas been found possible to include one or more enzymes in compositionsaccording to the invention and to provide sufficient boron in thecomposition both to protect the quat. biocide from deactivation by theenzyme, and to protect the quat. biocide from deactivation by anadditional protein (i.e. additional to the enzyme) and also to stabilisethe enzymes against being denatured by the quat. It may be that acomplex of the quat (e.g. with the protein) participates in reversiblyprotecting the enzyme. The enzymes may for example be proteolyticenzymes or selected from carbohydrases, esterases, hydrazes, amylases,proteases, catalases, lipases, amylases, cellulases, peroxidases,invertases, and the like together with mixtures thereof. The use ofproteases is preferred, and of subtisilin is highly preferred.

[0097] Surfactant

[0098] In preferred embodiments of the invention a surfactant ispresent. The surfactant is a non-ionic surfactant and it is highlypreferred that it be selected from alkoxylated alcohols or alkoxylatedphenol ethers or mixtures of them. Other semipolar nonionics such astrialkyl amine oxides may also be useful. Examples of alkoxylated phenolethers include octyl or nonyl phenol ether with varying degrees ofalkoxylation. 6-10 moles of ethylene oxide per mole of phenol ispreferred. The alkyl group can vary from C6-C16 The more highlypreferred are low alkoxylated nonionics having 6-25 moles of ethyleneoxide and/or propylene oxide per molecule

[0099] The alkoxylated alcohols include ethoxylated and propoxylatedC6-C16 alcohols with about 2-10 moles of ethylene oxide, or 1-10 and1-10 moles of ethylene and propylene oxide per mole of alcoholrespectively.

[0100] If amine oxides are used these may be mono-long chain, di-shortchain, trialkylamine oxides and can be ethoxylated or propoxylated, anexample is lauryl amine oxide, or cocoamidopropyldimethylamine oxide.

[0101] The quantity of surfactant is chosen so as to provide sufficientdetergency for soil removal, and will typically be in the range of from0.05% to 10% of the concentrate more preferably about 0.5% to 6% andmost preferably from 2%-4%

[0102] Treatment with the disinfectant cleaner herein described may befollowed by sterilisation using heat, chemicals, or any other suitablesterilizing system.

[0103] However if sterilisation (complete kill of viable organisms) isnot required, an adequate level of disinfection may be obtainable byrinsing with sterile water or other sterile solvent Hospital Grade Bdisinfection may be obtained merely by rinsing with sterile water afterthe treatment herein described.

[0104] The fact that certain monomeric quat. biocides do not require tobe rinsed even from food-contacting surfaces when applied at low levelsprovides an opportunity to formulate single-step cleaners/disinfectantsuseful on certain implements for dental use, denture cleaners and thelike.

[0105] The invention is herein described with particular reference toboron as the “activity protector” and quaternary ammonium biocides. Itmay be that not all enzyme stabilizer systems are effective as quat.biocide activity protectors, but those which are and are not effectivecan be determined by routine screening based upon the teaching hereof.

[0106] It will also be understood that while the invention has beendescribed with reference to the use of quat. biocides in the cleaningbath to prevent microorganism multiplication and protect staff, theconcept of including an effective biocide in the cleaning solution at alevel which is at least biostatic could be practiced by use of otherbiocidal systems, and such systems are within the scope of the inventionherein disclosed. The invention may be embodied in many forms which willbe apparent to those skilled in the arts of formulation, based upon theteaching herein contained.

[0107] Schedule 1

[0108] The TGA Disinfectant Test

[0109] This test method has been reproduced with the kind permission ofthe author and publisher from an original paper published in the“Australian Journal of Hospital Pharmacy”, Vol 8, No 4; 1978 (152-155).

[0110] 1. Principle

[0111] The method, as applied to Hospital Grade Disinfectants orSanitisers, is essentially that given by Kelsey & Maurer (1) for testingdisinfectant performance. It is set out in a form suitable forattachment to a regulatory minimum standard for disinfectants andantiseptics. For wider application of the test refer to supplementarynote A.

[0112] The disinfectant is tested at the dilution recommended by themanufacturer on the product label. The test consists of challenging thediluted disinfectant with bacterial inoculum, withdrawing a sample aftera given time and culturing the sample in a suitable recovery medium.After this sampling, the mixture is again challenged by a secondinoculum and after a second interval is again sampled for culturing. Thesample is passed or failed according to the extent of growth shown inthe two cultures sampled. The test may be performed with or without theaddition of sterile yeast as an organic soil. (Options B and Arespectively) or both, according to the use-situations advocated on thelabel of the product under test. TABLE 3 Selection of test parametersfor classes of disinfectant and antiseptic using the TGA DisinfectantTest. Test option for Organisms used resuspension of Number of Class ofproduct in the test centrifuged organisms challenges Inoculum densityDisinfectant - Ps. aeruginosa A (“clean” conditions) 2 2 × 10⁸-2 × 10⁹hospital grade: Pr. Vulgaris Sanitiser E. Coli B (“dirty” conditions) S.aureus Disinfectant - E. coli C 1 2 × 10⁸-2 × 10⁹ household or S. aureuscommercial grade Antiseptic Ps. aeruginosa D 1 1 × 10⁶-1 × 10⁷(excluding Pr. Vulgaris those for intact E. coli skin only) S. aureus

[0113] For Household Grade disinfectants, the first two organisms listedand the second challenge are omitted, while Option C (nutrient broth) isselected as the choice of simulated soil. For antiseptics, the secondchallenge is again omitted, while Option D (serum) is selected as thechoice of soil.

[0114] 2. Media

[0115] All media must be contained in capped glass containers. Wheremedia are stored, the containers must be sealed tightly or refrigerated.

[0116] 2.1 Sterile Hard Water

[0117] 2.1.1 Dissolve 0.304 g anhydrous calcium chloride and 0.065 ganhydrous magnesium chloride in glass-distilled water, and make up toone litre.

[0118] 2.1.2 Dispense into glass containers and sterilize by autoclavingat 121°±1° C. for 15 minutes.

[0119] 2.2 Yeast Suspension

[0120] 2.2.1 Weight 200 g of moist compressed baker's yeast. Cream bythe gradual addition of sterile hard water using a heavy glass rod forstirring. Decant the creamed portion into a flask, add more water to anylumpy residue remaining and repeat the creaming and decantation until noresidue remains and 500 ml of water has been used.

[0121] 2.2.2 Shake the contents of the flask vigorously and strainthrough a 100-mesh sieve, breaking down any remaining lumps.

[0122] 2.2.3. Add 500 ml sterile hard water, shake vigorously and adjustthe pH to 6.9-7.1 with 1N Sodium hydroxide.

[0123] 2.2.4 Transfer 50 ml, 100 ml or 200 ml of the yeast solution intoscrew-capped bottles.

[0124] 2.2.5 Autoclave at 121°±1° C. for 15 minutes and allow theautoclave to cool without releasing pressure. Store cold but notfreezing.

[0125] 2.2.6 Dry two Petri dishes to constant weight. Into each, pipette25 ml of sterilised yeast suspension, and dry to constant weight at 100°C. Calculate the average solids content of the suspension.

[0126] 2.2.7 Before use, pipette 25 ml of the sterilised yeastsuspension into a beaker. Determine the pH using the glass electrode,and determine the volume of 1N sodium hydroxide solution needed toadjust the pH to within the range 6.9 to 7.1.

[0127] 2.2.8 Immediately before use, add to each bottle of sterilisedyeast, a volume of sterile hard water and a volume of 1N sodiumhydroxide calculated to adjust the concentration of dry yeast to 5.0%and the pH to within the range 6.9-7.1. Discard prepared yeast 3 monthsafter preparation.

[0128] 2.3 Medium for Growth of Test Organisms

[0129] 2.3.1 Prepare a 10% w/v dextrose solution in distilled water, andsterilise by autoclaving at 121°±1° C. for 15 minutes. Cool to roomtemperature.

[0130] 2.3.2 Prepare Wright and Mundy medium following the author'sprocedure (2) or from a commercial product of the same composition (NoteB) and sterilise by autoclaving at 121°±1° C. for 15 minutes. Cool toroom temperature.

[0131] 2.3.3 To each litre of Wright and Mundy medium prepared in 2.3.2add 10 ml sterile dextrose solution prepared in 2.3.1.

[0132] 2.3.4 Aseptically dispense in either 10 ml or 15 ml amounts, aspreferred.

[0133] 2.3.5 This medium is referred to as Wright and Mundy dextrosemedium.

[0134] 2.4 Recovery Medium

[0135] 2.4.1 Prepare nutrient broth as follows or from a commercialproduct of the same composition (Note B):

[0136] Add the following to 970 ml of water and dissolve by heating.Beef Extract Powder 10 g Peptone 10 g Sodium Chloride  5 g Adjust the pHto 8.0-8.4 using 1N Sodium Hydroxide. Boil for 10 minutes and filter.Cool.

[0137] 2.4.2 To each litre of nutrient broth solution prepared in 2.4.1add 30 g polysorbate 80 (Note B).

[0138] 2.4.3 Adjust pH to 7.2-7.4, using 1N Sodium hydroxide.

[0139] 2.4.4 Autoclave at 121°±1° C. for 15 minutes, and immediatelyshake well to disperse the polysorbate 80.

[0140] 2.4.5 Dispense aseptically in 10 ml amounts into sterile cappedglass tubes.

[0141] 3. Test Inoculation

[0142] 3.1 Test Organisms

[0143] The following 4 organisms are to be used, except whereprescribed. Pseudomonas aeruginosa NCTC 6749 Proteus vulgaris NCTC 4635Escherichia coli NCTC 8196 Staphylococcus aureus NCTC 4163

[0144] 3.2 Preparation of Inoculation

[0145] 3.2.1 Incubate the contents of an ampoule of freeze-dried cultureovernight at 37°±1° in Wright and Mundy dextrose medium.

[0146] 3.2.2 Inoculate the incubated culture onto nutrient agar slopesin McCartney bottles. Store for up to 3 months at 4°±1° C.

[0147] 3.2.3 At a suitable period before the test is to be conducted,sub-culture from an agar slope into 10 ml or 15 ml quantities of Wrightand Mundy dextrose medium. Incubate at 37°±1° C. for 24±2 hours.

[0148] 3.2.4 Sub-culture from the medium in 3.2.3 into fresh medium,using an inoculating loop of 4 mm in diameter. Incubate at 37°±1° C. for24±2 hours.

[0149] 3.2.5 Repeat step 3.2.4 daily. For the test procedure use onlythose cultures which have been sub-cultured at least 5, and not morethan 14 times.

[0150] 3.2.6 Filter test cultures of P. aeruginosa and S. aureus throughsterile Whatmans No. 4 filter paper.

[0151] 3.2.7 Centrifuge all test cultures until cells are compact, andremove supernatant with a Pasteur pipette.

[0152] 3.2.8 Resuspend test organisms in the original volume of liquid(i.e. 10 ml or 15 ml), and shake for 1 minute with a few sterile glassbeads.

[0153] 3.2.8.1 For Option A, resuspend in sterile hard water.

[0154] 3.2.8.2 For Option B, resuspend in a mixture of 4 parts yeastsuspension (prepared as in 2.2) to 6 parts sterile hard water.

[0155] 3.2.8.3 For Option C, resuspend in nutrient broth (prepared as in2.4.1 and 2.4.3 and sterilised by autoclaving).

[0156] 3.2.8.4. For Option D, resuspend in sterile hard water; dilutetwice 1+9 in sterile hard water; then add 8 ml of the last dilution to 2ml sheep serum previously inactivated at 56° C. for 20 mins andsterilised by filtration.

[0157] 3.3 Enumeration of Inoculation

[0158] Immediately before testing, sample the resuspended inoculum andenumerate using 10-fold dilutions in quarter-strength Ringer's solutionand the pour-plate technique. The number subsequently counted mustrepresent not less than 2×10⁸ or more than 2×10⁹ organisms permillilitre (or 1×10⁸-1×10⁷ using Option D) or the test is consideredinvalid. Retain tube containing 10⁻⁷ dilution for use in controls (7.3and 7.4).

[0159] 4. Disinfectant Dilutions

[0160] Quantitatively dilute a sample of the disinfectant to thespecified extent, using sterile hard water as diluent. Use not less than10 ml or 10 g of sample for the first dilution, and not less than 1 mlof any dilution to prepare subsequent dilutions. Make all dilutions inglass containers on the day of testing. The glass containers must betwice rinsed in glass-distilled water, and sterilised.

[0161] 5. Temperature

[0162] Where air-conditioning does not maintain test solutions at 21°±1°C., hold the containers in which the test is to be carried out in awaterbath at this temperature.

[0163] 6. Test Procedure

[0164] Perform the following test using each of the four test organisms(3.1) except where the Standard directs otherwise. It is not necessaryto test with all organisms simultaneously.

[0165] 6.1 Add 3 ml of diluted disinfectant to a capped glass container.

[0166] 6.2 Start a timing device. Immediately inoculate disinfectantwith 1 ml of culture (prepared in 3.2) and mix by swirling.

[0167] 6.3 At 8 minutes, subculture one drop (0.02 ml±0.002 ml) intoeach of 5 tubes containing recovery broth. To ensure delivery of 0.02 mlinto the first tube of recovery broth at exactly 8 minutes, it will benecessary to withdraw a suitable amount from the disinfectant test mixshortly beforehand. This must be immediately preceded by vortexing.Surplus sample must be returned to the test mix (See Note D).

[0168] 6.4 Except where prescribed, at 10 minutes, inoculatingdisinfectant with a further 1 ml of culture, and mix by vortexing.

[0169] 6.5 Except where prescribed, at 18 minutes, proceed as in 6.3.

[0170] 6.6 Mix the contents of all tubes of recovery broth by vortexing.Incubate at 37°±1° C. for 48±2 hours.

[0171] 6.7 Examine for growth and record results.

[0172] 6.8 For each test organism repeat steps 6.1-6.7 on each of 2subsequent days, using fresh disinfectant dilution and a freshlyprepared bacterial suspension.

[0173] 7. Controls

[0174] 7.1 Recovery broth contamination Incubate one uninoculated tubeof recovery broth at 37°±1° C. for 48±2 hours and examine for growth. Ifgrowth occurs, the test is considered invalid due to contamination ofthe recovery broth.

[0175] 7.2 Disinfectant Contamination

[0176] To 1 tube of recovery broth, add 0.02 ml of diluted disinfectant.Incubate at 37°±1° C. for 48±2 hours. If growth occurs, the test isconsidered invalid. Growth in 7.2 but not 7.1 indicates contamination ofthe disinfectant test solution.

[0177] 7.3 Fertility Test

[0178] To 1 tube of recovery broth, add 1.0 ml of the 10⁻⁷ dilutionretained in 3.3. Incubate at 37°±1° C. for 48±2 hours and examine forgrowth. If no growth occurs, the test is considered invalid.

[0179] 7.4 Inactivator Efficacy

[0180] To 1 tube of recovery broth, add 0.02 ml of diluted disinfectantand 1.0 ml of the 10⁻⁷ dilution retained in 3.3. Incubate at 37°±1° C.for 48±2 hours, and examine for growth. If no growth occurs, the test isconsidered invalid. Growth in 7.3 but not in 7.4 indicates inadequateinactivation of the disinfectant.

[0181] 8. Procedure in Case of Invalid Controls

[0182] When any control renders the test invalid, the test is to berepeated. Fresh recovery broth is to be used if growth occurred incontrol 7.1 or if no growth occurred in controls 7.3 or 7.4.

[0183] Should disinfectant contamination be indicated by control 7.2 onboth occasions, the disinfectant is considered to fail the test. Shouldinadequate inactivation of the disinfectant be indicated by control 7.4on both occasions, the test is considered invalid (Note C).

[0184] 9. Results

[0185] The dilution test passes the test if there is no apparent growthin at least two out of the five recovery broths specified in 6.3 and noapparent growth in at least two of the five recovery broths specified in6.5 on all three occasions, using all four organisms.

10. REFERENCES

[0186] (1) Kelsey, J. C. and Maurer Isobel, M. Pharmaceutical Journal(UK) 213: 528-530, (1974).

[0187] (2) Wright Eleanore, S. and Mundy, R. A. Journal of Bacteriology80: 279-280, (1960).

[0188] 11. Supplementary Notes

[0189] A. For investigational, developmental or comparative purposes, itwill be useful to add a third challenge thus performing a true capacitytest, and to test at dilutions above and below the prescribed dilution.In such cases, Kelsey & Maurer's recommendations regarding the timingand organisation of the test should be carefully consulted.Abbreviations of the test may be considered for the routine test ofproduction batches.

[0190] B. Wright & Mundy medium is commercially available as “BactoSynthetic Broth”, A.O.A.C. Code No. 0352 (Difco Ltd.). The nutrientbroth to be used is available as “Nutrient Broth—No. 2” (Oxoid Ltd.).

[0191] C. Where inadequate inactivation is indicated, investigationsshould be conducted to find an effective inactivator. Refer Mackinnon,I. H. J. Hyg (London) 73: 189-195, (1974).

[0192] D. The Oxford P-7000 sampler system with disposable plastic tipsis recommended for the withdrawal of samples for subculturing. Schedule2 Acceptable Common Names Descriptive Name Common Names SterilantSterilant Instrument Grade - Instrument grade - High level disinfectanthigh level disinfectant or High Level Instrument Disinfectant orInstrument Disinfectant - high level or High Level Instrument GradeDisinfectant or High Level Disinfectant or Instrument Grade DisinfectantInstrument Grade - Instrument Grade - intermediate level intermediatelevel disinfectant or Intermediate Level disinfectant Instrument gradeDisinfectant, or Intermediate Level Instrument Disinfectant orIntermediate Level Disinfectant Instrument Grade - low InstrumentGrade - low level level disinfectant disinfectant or Low LevelInstrument Grade Disinfectant, or Low Level Disinfectant, or InstrumentGrade Disinfectant - low level Hospital grade disinfectantDisinfectant - hospital grade (see Surface spray below if Hospital GradeDisinfectant primarily for use as a spray) Household/Commercial gradeDisinfectant - household grade, or disinfectant (see SurfaceDisinfectant - commercial grade, or spray below if primarily HouseholdGrade Disinfectant, or for use as a spray) Commercial Grade DisinfectantSurface spray disinfectant Surface spray disinfectant - hospital grade,or Surface spray disinfectant - household grade, or Surface spraydisinfectant - commercial grade Antibacterial clothes Antibacterial(together with a word or preparation words indicating the nature of theproduct) Sanitary fluid Sanitary fluid Sanitary powder Sanitary powderSanitiser Sanitiser, or Sanitising Solution, or Antibacterial (togetherwith a word or words indicating the nature of the product)

What is claimed is:
 1. A method for reducing the risk to health careworkers when cleaning a medical instrument contaminated with an organicload including protein, said method comprising the step of treating thecontaminated instrument with a liquid composition comprising a protease,a monoquaternary ammonium disinfectant and an activity protector, theactivity protector being of a kind and in a concentration selected sothat the liquid provides hospital grade disinfection (as herein defined)in the presence of said protease and said load.
 2. A method according toclaim 1 further comprising the step of sterilizing the instrument.
 3. Amethod according to claim 1 further comprising the step of rinsing theinstrument.
 4. A liquid composition for use in cleaning a medicalinstrument contaminated with an organic load comprising a protein, saidcomposition comprising: a protease a mono quaternary ammonium biocide;and an activity protector of a kind and in a concentration selected sothat the liquid provides hospital grade disinfection (as herein defined)in the presence of said protease and said load.
 5. A compositionaccording to claim 4 wherein the composition further comprises one ormore enzymes selected from the group consisting of proteolytic enzymes,carbohydrases, esterases, hydrazes, amylases, catalases, lipases,amylases, cellulases, peroxidases, invertases and mixtures thereof.
 6. Acomposition according to claim 4 wherein the activity protector is orcomprises a micelle formation inhibitor.
 7. A composition according toclaim 4 wherein the monoquaternary biocide is a monomeric quaternaryammonium antimicrobial compound selected from the group having a generalformula:

wherein R′, R″, R′″, R″″ are alkyl radicals that may be the same ordifferent, substituted or unsubstituted, branched or unbranched, andcyclic or acyclic, and X is any anion.
 8. A composition according toclaim 7 wherein X is chlorine, bromine or other halogen.
 9. Acomposition according to claim 4 wherein the quaternary biocide isselected from the group consisting of mono-long-alkyl chain, tri-shortchain, tetralkyl ammonium compounds; di-long-chain, di-short chaintetralkyl ammonium compounds and mixtures thereof.
 10. A compositionaccording to claim 4 wherein the quaternary biocide is selected from thegroup consisting of monoalkyltrimethyl ammonium salts,monoalkyldimethylbenzyl compounds, dialkylbenzyl compounds andQuaternary gluconates.
 11. A composition according to claim 4 whereinthe biocide is selected from the group consisting of C8 to C22 dimethylbenzyl ammonium chloride, C8-C22 dimethyl ethyl benzyl ammonium chlorideand di-C6-C20 alkyl dimethyl ammonium chloride.
 12. A compositionaccording to claim 4 wherein the quaternary biocide is a benzyl dimethylammonium halide.
 13. A composition according to claim 4 wherein thebiocidal efficacy of the quaternary biocide is protected by an “activityprotector” selected from the group consisting of “enzyme stabilizers”,“enzyme stabilizing systems”, “micelle formation modifiers andinhibitors”, and combinations thereof.
 14. A composition according toclaim 13 wherein the biocidal efficacy of the quaternary biocide isprotected by one or more enzyme stabilizers and stabilizer enhancersselected from the group consisting of boron compounds, polyols,formates, calcium ions, polyfunctional amino compounds, phosphates,citrates, sulphates and sequestering agents.
 15. A composition accordingto claim 4 wherein a stabilizer is selected from boric acid, boricoxide, borax, or sodium ortho-, meta-, or pyro-borate and perborates.16. A composition according to claim 4 wherein a stabilizer comprisingsodium tetraborate.
 17. A composition according to claim 13 wherein thebiocidal efficacy of the quaternary biocide is protected by a boroncompound and further comprising a polyol having from 2 to 6 hydroxylgroups.
 18. A composition according to claim 14 wherein the polyol isselected from the group consisting of ethylene glycol, propylene glycol1,2 propanediol, butyleneglycol, glycerol, mannitol, sorbitol,erythritol, glucose, fructose and lactose.
 19. A composition accordingto claim 17 wherein the polyol is selected from the group consisting ofglycerol, mannitol, sorbitol, erythritol, glucose, fructose and lactose.20. A composition according to claim 13 wherein the biocidal efficacy ofthe quaternary biocide is protected by a micelle immiscible solvent. 21.A composition according to claim 13 wherein the micelle immisciblesolvent is selected from the group consisting of C1-C6 alkanols, C1-C6diols, C3-C24 alkylene glycol ethers, alkylene glycol alky ethers,borates, lactates, citrates, tartrates and mixtures thereof.
 22. Acomposition according to claim 21 wherein the solvent comprises di(propylene glycol) methyl ether (“DPM”).
 23. A composition according toclaim 4 wherein the “activity protector” comprises boron ions.
 24. Acomposition according to claim 4 further comprising DPM.
 25. Acomposition according to claim 4 further comprising a non ionicsurfactant.
 26. A composition according to claim 4 being a shelf stableliquid disinfectant concentrate composition containing at least 1% byweight of a quaternary biocide and capable of dilution with 20 parts ofwater to 1 part of concentrate, the diluted solution exhibiting a MICafter 24 hrs in the presence of up to 2% of tryptone (or the proteinequivalent thereof) which is less than the MIC of a solution of the sameconcentration of the same quaternary biocide in distilled water in thepresence the same amount of the protein.
 27. A composition according toclaim 26 wherein the protease is subtilisin.
 28. A composition accordingto claim 26 when diluted by more than 20 parts of water to 1 ofconcentrate.
 29. A composition according to claim 26 when diluted bymore than 100 parts of water to 1 of concentrate.
 30. A compositionaccording to claim 26 when diluted by more than 200 parts of water to 1of concentrate.
 31. A method for cleaning a surgical instrumentcomprising the steps of immersing the instrument in a solution accordingto claim 4, and subsequently sterilizing the instrument.
 32. A methodfor cleaning a surgical instrument comprising the steps of immersing theinstrument in a solution according to claim 4, and subsequently rinsingwith a sterile solvent.